Genetic information storage and processing rely on just two polymers, DNA and RNA. Whether their role reflects evolutionary history or fundamental functional constraints is unknown. The nucleic acids DNA and RNA provide the molecular basis for all life through their unique ability to store and propagate information. To better understand these singular properties and discover relevant parameters for the chemical basis of molecular information encoding, nucleic acid structure has been dissected by systematic variation of nucleobase, sugar and backbone moieties.
Prior art studies have revealed the profound influence of backbone, sugar and base chemistry on nucleic acid properties and function. Crucially, only a small subset of chemistries allows information transfer through base pairing with DNA or RNA, a prerequisite for crosstalk with extant biology. However, base pairing alone cannot conclusively determine the capacity of a given chemistry to serve as a genetic system, as hybridization need not preserve information content. A more thorough examination of candidate genetic polymers' potential for information storage, propagation and evolution requires a system for replication which would allow a systematic exploration of the informational, evolutionary and functional potential of synthetic genetic polymers and open up applications ranging from biotechnology to material science.
In principle, informational polymers can be synthesized and replicated chemically with advances in the non-enzymatic polymerization of mononucleotides and short oligomers enabling model selection experiments. Nevertheless, chemical polymerization remains relatively inefficient, which is a problem in the art.
On the other hand, enzymatic polymerization has been hindered by the stringent substrate selectivity of polymerases. Despite progress in understanding the determinants of polymerase substrate specificity and in engineering polymerases with expanded substrate spectra, most unnatural nucleotide analogues are poor polymerase substrates at full substitution, both as nucleotides for polymer synthesis and as templates for reverse transcription. Notable exceptions are 2′OMe-DNA and TNA. 2′OMe-DNA is present in eukaryotic rRNAs, is well-tolerated by natural reverse transcriptases (RTs) and has been shown to support heredity and evolution at near full substitution. TNA allowed polymer synthesis and evolution in a three letter system but only limited reverse transcription. Thus, polymerase substrate specificity remains a significant drawback to progress in this area.
WO2011/135280 describes certain polymerases. This document describes a polymerase which is capable of making an RNA polymer from a DNA polymer template. This polymerase is termed TGK. This polymerase comprises mutations Y409G and E664K. The TGK polymerase is described as the first primer dependent thermostable RNA polymerase engineered from a DNA scaffold. The TGK polymerase can synthesise a tRNA gene in less than a minute and can synthesise a 1.7 kb luciferase gene in only one hour. Thus, the TGK polymerase is described as a very efficient RNA polymerase.
WO2011/135280 describes polymerase D4, which is a processive, high fidelity RNA polymerase. The D4 polymerase is based on the TgoT polymerase but with a number of mutations, in particular eight mutations in the region of the thumb domain (motif 10A). The amino acid and nucleotide sequences of the TgoT-derived polymerase D4 is shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. In addition, the Y409N mutation is added to the D4 polymerase to make the polymerase D4N3. This D4N3 polymerase exhibits a striking ability to processively synthesize RNAs up to 87 nucleotides in length.
WO2011/135280 discusses mutation of the E664 residue of TgoT. It is mentioned that E664 may be mutated to E664K, or E664Q, or a small number of other resides as shown in the table at the top of page 51 of WO2011/135280. It was concluded in WO2011/135280 that the E664Q mutation is both necessary and sufficient for processive RNA synthesis (page 80 lines 21 to 22 of WO2011/135280).
The discovery that ANA and FANA in hybrid duplexes with RNA activate RNaseH led to interest in their use for siRNA and FANA in particularly effective at gene knockdown. Furthermore, FANA is stable to chemical (acid or base) hydrolysis, serum resistant, can improve the binding of DNA aptamers, stabilizes G-quadruplexes, hybridizes with RNA more tightly than RNA:RNA and can silence genes more effectively than native (RNA) siRNA. As a result FANA is of interest as a backbone for aptamer generation.
No polymerases are known in the art which are capable of producing arabino nucleotide polymers, such as ANA polymers or FANA polymers.
The present invention seeks to overcome problems associated with the prior art.